Project Summary Glioblastoma (GBM) is one of the most lethal incurable human diseases. Even with aggressive therapies including surgical resection followed by radiotherapy and chemotherapy using temozolomide (TMZ), the median survival for GBM patients is only 14 months. In fact, GBM cancer cells are highly infiltrative and comprise a sub- population of glioma stem cells (GSCs) with tumorigenic properties regulated by the tumor microenvironment, and often resistant to chemotherapy and irradiation treatments. The remaining GSCs can then enter an active state of self-renewal, and asymmetric division that recapitulates the heterogeneous tumor. As a result, all treated GBM patients will experience tumor recurrence and subsequent surgeries and toxic radiotherapy and chemotherapy regimens are harmful for the patient and often remain insufficient. There is therefore an urgent need for new therapeutic drug to target GSCs and treat this devastating disease. Glioblastoma resistance to TMZ correlates with the expression of the gap junction protein Connexin43 (Cx43), a protein which enables communication between cells, and increased levels of Cx43 are observed in GSCs. The function of Cx43, is not limited to forming channels for the passage of ions and small molecules between cells, but also participates in cell proliferation, migration and apoptosis. Therefore, targeting Cx43 activity holds promise to treat GBM and prevent tumor recurrence. In this proposed research we use a novel Cx43 mimetic peptide named JM2 (juxtamembrane 2) that encompasses the microtubule binding sequence of Cx43. Our preliminary data show that JM2 alters Cx43 binding to microtubule, decreases the formation of Cx43 gap junctions, and inhibits cell- cell communication in GSCs. Most importantly, we reveal the therapeutic potential of JM2 in decreasing GSC survival in vitro and in vivo. With the goal of developing a new therapy based upon JM2 to target GSCs in GBM, our overall objective is to generate JM2-loaded polyanhydride biodegradable nanoparticles (JM2-NPs) for sustained delivery of JM2 to GSCs. We will use high-resolution microscopy techniques including stochastic optical reconstruction microscopy (STORM) and biochemistry assays to analyze the effect of JM2-NPs in GSCs derived from human primary GBM cells freshly isolated from dissected patient tumor. Finally, we will assess the therapeutic effect of JM2-NPs on GSCs ex vivo using a three dimensional patient GBM-derived organoid model, and in vivo using an orthotopic GBM mouse model. These results will validate the potent effect of JM2 peptide for treatment of high Cx43/chemoresistant GSCs and present a therapeutic opportunity to prevent GBM tumor recurrence. The proposed research is significant because this innovative approach will not only allow us to develop novel therapies for lethal GBM but also will lay foundation on potential clinical trials in newly diagnosed GBM patients in the near future. Finally, our new JM2-loaded nanoparticles may be scalable to other CNS diseases that could benefit from targeting Cx43-microtubule interaction.